1. 17May06KL Vadheim Lecture 31 Diphtheria, Tetanus, Pertussis MedCh 401 Lecture 3

2. 17May06KL Vadheim Lecture 32 Adjuvant Substances that enhance the immune response Two categories: –vehicles –immunomodulators

3. 17May06KL Vadheim Lecture 33 Adjuvants functioning as vehicles I Human use: –Alum compounds Aluminum hydroxide and phosphate the only licensed adjuvants in U.S. –MF59 Oil and water emulsion Marketed in Europe

4. 17May06KL Vadheim Lecture 34 Adjuvants functioning as vehicles II Animal use: –Freund’s Complete Adjuvant (CFA) dessicated Mycobacterium butyricum, mineral oil and an emulsifying agent, mannide monooleate causes potentially secere local inflammatory lesions, chronic granulomas, abscesses, and tissue sloughs. Injected into the murine footpad, it can cause chronic lameness and arthritis; injected intraperitoneally, it can cause peritonitis –Freund’s Incomplete Adjuvant Mineral oil and Mannide monooleate Fewer side effects, adequate for boosting

5. 17May06KL Vadheim Lecture 35 Immunomodulatory Adjuvants Purified Protein Derivative (PPD) Lipopolysaccharide (LPS; bacterial endotoxin) Lipid A - lipid portion of LPS Cholera toxin B subunit CpG

6. 17May06KL Vadheim Lecture 36 Diphtheria vaccine Detoxified bacterial, protein toxin Injectable, IM administration Toxigenic Corynebacterium diphtheriae (infected with  phage) Primarily a childhood disease

7. 17May06KL Vadheim Lecture 37 Diphtheria Transmission Person-to-person by respiratory droplet No animal reservoir

8. 17May06KL Vadheim Lecture 38 Manufacturing Process Toxigenic strain of C. diphtheriae grown in Fenton medium with a bovine extract After suitable growth, toxin purified from cells by centrifugation Toxoided by incubation with formaldehyde for several weeks Concentrated with ultrafiltration Purified by precipitation, dialysis and sterile filtered Adsorbed onto aluminum hydroxide, Al(OH) 3

9. 17May06KL Vadheim Lecture 39 Excellent Vaccine Efficacy Mortality –1860-189746-196/100,000 people –Case fatality rates >50% during outbreaks –1920 15/100,000 people Cases –1971 - 1981 -1,288 in U.S. –1980 - 1995 -41 –occasional in developing countries and Native populations in U.S. and Canada

10. 17May06KL Vadheim Lecture 310 Tetanus Vaccine Detoxified bacterial, protein toxin Clostridium tetani Injectable dosage form IM

11. 17May06KL Vadheim Lecture 311 Tetanus Transmission Not a communicable disease The only vaccine-preventable infection that is not communicable Disease acquired through exposure to bacterial spores in the environment –inoculation of bacterial spores into body by puncture or deep cut

12. 17May06KL Vadheim Lecture 312 Transmission II Neonatal tetanus most common worldwide Case fatality increases with age (~50% for 80+ years of age) Disease reservoirs –Soil –Animal feces

13. 17May06KL Vadheim Lecture 313 Tetanus toxins Tetanolysin - possible role in establishing infection at inoculation site Tetanospasm –accumulates intracellularly during log-phase growth –released into medium upon autolysis –Minimum human lethal dose ~ 2.5 ng/kg

14. 17May06KL Vadheim Lecture 314 Tetanus disease Tetanospasms –localized - spasm of muscles close to site of injection; weeks to months duration; rare but may precede generalized symptoms –generalized - 80% of cases Complications of the spasms: –fractures of the long bones and vertebrae –asphyxia from glottic obstruction

15. 17May06KL Vadheim Lecture 315 Nervous system effects Toxin travels up nerve endings by intra- axonal transport Gains entry to neuromuscular junctions by binding to gangliosides Interferes with release of neurotransmitters from presynaptic inhibitory fibers Excitatory reflexes multiply unchecked, causing spasms

16. 17May06KL Vadheim Lecture 316 Tetanus Toxin-mediated disease Symptoms may progress clinically despite use of parenteral antibiotics Treatment: –hyperimmune serum –supportive care Recovery may depend on development of new functional nerve connections

17. 17May06KL Vadheim Lecture 317 Manufacturing Process Growth of C. tetani in modified Latham broth in fermenters Harvest extracellular toxin by filtration Purify Detoxify with formaldehyde for ~3 weeks Adsorb with Alum adjuvant Diafiltration

18. 17May06KL Vadheim Lecture 318 Pertussis (Whooping Couth) Bordetella pertussis Whole cell and acellular vaccines –traditional and recombinant Injectable dosage form IM

19. 17May06KL Vadheim Lecture 319 Pertussis Transmission Person-to-person Respiratory droplets

20. 17May06KL Vadheim Lecture 320 Pertussis (whooping cough) Killed Whole cell - –old, not licensed in U.S. or Europe –still used in developing countries –relatively cheap Acellular (aP) - –currently licensed in U.S., Japan and Europe –some are recombinant –expensive

21. 17May06KL Vadheim Lecture 321 B. pertussis virulence factors Pertussis toxin (PT) Filamentous hemagglutinin (FHA) Fimbriae (fimbrial agglutinogens) (Fim) Pertactin (PRN) Adenylate cyclase Tracheal cytotoxin Dermonecrotic or heat-labile toxin BrkA Endotoxin (LPS)

22. 17May06KL Vadheim Lecture 322 Vaccine Efficacy 1914 (Pre-vaccine) –~270,000 cases, 10,000 deaths annually –killed >5 of every 1000 children born in U.S. 1976 - 1,010 cases 1996 - 7,796 2002 –8,296 cases –3.01/100,000 pop.

23. 17May06KL Vadheim Lecture 323 Vaccine History 1914 First whole-cell vaccine (P) 1948 DTP 1991 - 2002 DTaP for children 2005 Tdap for adults 19-64

24. 17May06KL Vadheim Lecture 324 Manufacturing B. pertussis cultures grown in modified Stainer-Scholte broth Acellular antigens: –purified from culture medium (PT and FHA) or –extracted from cells (PRN, FIM) Formaldehyde and/or glutaraldehyde detoxification of PT Aluminum adjuvants

25. 17May06KL Vadheim Lecture 325 Manufacturing II Novartis-Behring (Chiron) - Recombinant –Genetically detoxified PT –Two other components –Not available in U.S. yet

26. 17May06KL Vadheim Lecture 326 DTaP Vaccine Formulations

27. 17May06KL Vadheim Lecture 327 Licensed DTaP Vaccines